Human parainfluenza virus (HPIV) serotypes 1, 2, and 3 are significant causes of severe respiratory tract disease in infants and young children. The HPIVs are enveloped, non-segmented, single-stranded, negative-sense RNA viruses belonging to subfamily Paramyxovirinae within the Paramyxoviridae family. These serotypes can be further classified as belonging to either the Respirovirus (HPIV1 and HPIV3) or Rubulavirus (HPIV2) genus and are immunologically distinct in that primary infection does not result in cross-neutralization or cross-protection. The HPIV genome encodes three nucleocapsid-associated proteins including the nucleocapsid protein (N), the phosphoprotein (P) and the large polymerase (L) and three envelope-associated proteins including the internal matrix protein (M) and the fusion (F) and hemagglutinin-neuraminidase (HN) transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and are the major viral protective antigens. In addition, the P gene encodes the accessory protein(s) C (HPIV1), V (HPIV2), and C, D, and possibly V (HPIV3). The HPIVs cause respiratory tract disease ranging from mild illness, including rhinitis, pharyngitis, and otitis media, to severe disease, including croup, bronchiolitis, and pneumonia. HPIV1, HPIV2 and HPIV3 have been identified as the etiologic agents responsible for 6.0%, 3.3% and 11.5%, respectively, of hospitalizations of infants and young children for respiratory tract disease. Together these viruses account for approximately 18% of all pediatric hospitalizations due to respiratory disease. Licensed vaccines are currently not available for any of the HPIVs. The major goal of this project is to develop live attenuated vaccines against all three serotypes. Candidate vaccine viruses are recovered from cDNA using reverse genetic systems described in previous reports. This provides the means to develop well-defined live vaccines. Based on previous work, we already have multiple HPIV3 vaccines in clinical trials. Thus, this report focuses on HPIV1 and HPIV2. HPIV1: A novel HPIV1 mutant, rHPIV1-C+P, was generated in which the overlapping open reading frames (ORFs) encoding the C and P proteins were placed in separate genes to make it possible to introduce mutations into one protein without affecting the other. Infectious rHPIV1-C+P was readily recovered and replicated as efficiently as wild-type (wt) HPIV1 in vitro and in African green monkeys (AGMs). rHPIV1-C+P provided a useful backbone for recovering an attenuated P/C gene deletion mutation, (del84-85), which was previously unrecoverable, likely due to detrimental effects of the deletion on the function of the P protein. rHPIV1-C(del84-85)+P and an additional mutant, rHPIV1-C(del169-170)+P, were found to replicate to similar titers in vitro and to activate the type I IFN and apoptosis responses to a similar degree as rHPIV1-C+P. rHPIV1-C(del84-85)+P was highly attenuated in AGMs, immunogenic, and effective in protecting AGMs against challenge with wt HPIV1, and will be investigated further as a candidate HPIV1 vaccine. We further sought to understand the role of the C proteins in apoptosis. HPIV1 was modified to create rHPIV1-P(C-), a virus in which expression of the C coding sequence was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C-) was readily recovered from cDNA, indicating that the nested set of four C proteins is not essential for virus replication. rHPIV1-P(C-) replicated in vitro as efficiently as wt HPIV1 early during infection, but its titer subsequently decreased coincident with the onset of extensive apoptosis not observed with wt HPIV1. The apoptosis was initiated by activation of both the intrinsic (caspase 9) and extrinsic (caspase 8) pathways. Contrary to expectations, this heightened apoptosis does not appear to be the cause of the reduction in viral replication, which remains unexplained. HPIV2: In wt HPIV2, a single gene (the P/V gene) encodes both the P protein and the accessory cysteine-rich V protein. The two proteins share the same N-terminal sequence but have different C-terminal domains due to frame-shifting (called RNA editing) by the viral polymerase. In model paramyxoviruses, V inhibits the cellular interferon (IFN) response, in addition to performing other functions that are less well defined. We mutated the RNA editing signal to create an HPIV2 mutant (rHPIV2-Vko) in which the P/V gene expressed only the P protein. Loss of expression of the V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. The rHPIV2-Vko virus, unlike wt HPIV2, strongly induced type I IFN and permitted signaling through the IFN receptor, leading to the establishment of a robust antiviral state. rHPIV2-Vko infection induced extensive syncytia and caused dramatic cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-Vko was highly restricted in the upper and lower respiratory tract (URT and LTR) of AGMs and was not detected in differentiated primary human airway epithelial (HAE) cultures, suggesting that the V protein is essential for efficient replication of HPIV2 in vivo and in HAE cultures in vitro. The high degree of restriction of rHPIV2-Vko in AGMs and in HAE cultures suggests that this mutant is over-attenuated and would not be suitable as a live attenuated virus vaccine, but will be useful to study the function of V during HPIV2 infection. We next characterized wt HPIV2 infection in the HAE in vitro model. We found that the virus replicates to high titer, is shed only apically, targets ciliated cells, and induces minimal cytopathology. Next, we used the HAE model to evaluate a live-attenuated vaccine candidate that was described in previous reports and is currently in clinical trials. This candidate, called rHPIV2-V94(15C)/948L/del1724, contains both temperature sensitive (ts) and non-ts attenuating mutations and was previously found to be restricted in replication in the URT and LRT of AGMs and to be protective against wt HPIV2 challenge. This mutant was reduced in replication by more than 30-fold compared to wt HPIV2 in HAE cultures at 32C and exhibited little productive replication at 37C, reflecting a restriction of replication similar to that in the cooler URT and warmer LRT of AGMs . These data indicate that the HAE model provides a convenient experimental system for examining the cell tropism, cytotoxicity, and attenuation phenotypes of HPIV2 vaccine candidates as well as for characterizing the innate host responses of human airway epithelial tissues to infection. Since clinical trials are the only true tests of vaccine safety and efficacy, the results from this study encourage continued evaluation of this vaccine candidate in clinical trials. As noted, the N-terminal domains of the P and V proteins are encoded by the same ORF, and their C-terminal domains by overlapping ORFs. In order to manipulate either of these proteins without affecting the other, we created an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Delta122-127. In contrast to wt HPIV2, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor wt HPIV2, induced significant amounts of IFN-beta in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Delta122-127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited.